One of the primary goals of plant genetic engineering is to obtain plants having improved characteristics or traits. These characteristics or traits include virus resistance, insect resistance, herbicide resistance, enhanced stability and improved nutritional value, to name a few. Recent advances in genetic engineering have enabled the incorporation of preselected genes into plant cells to impart the desired qualities to the plant of choice. The introduced gene, i.e., "transgene," is then expressed in the cells of the regenerated plant, so that the plant will exhibit the trait or characteristic encoded by the transgene.
To express a transgene in a plant cell, the proper regulatory signals must be present and in the proper location with respect to the transgene. These regulatory signals generally include a promoter region, a 5' non-translated leader sequence and a 3' polyadenylation sequence. The promoter region influences the rate at which the RNA product of the transgene, and resultant protein product of the transgene, is made. Promoter activity also can depend on the presence of several other cis-acting regulatory elements which, in conjunction with cellular factors, determine strength, specificity, and transcription initiation site (for a review, see Zawel and Reinberg, Curr. Opin. Cell Biol., 4, 488 (1992)). Strong promoters are able to direct RNA synthesis at a higher rate relative to weak promoters. Constituitive promoters direct RNA production in many or all cell types.
The cauliflower mosaic virus 35S promoter (CaMV35S) is a strong, constitutive promoter in plants (Odell et al., Nature, 313, 810 (1985); Jensen et al., Nature. 321, 669 (1986); Jefferson et al., EMBO J., 6, 3901 (1987); Kay et al., Science, 236, 1299 (1987); Sanders et al., Nucl. Acids Res., 4, 1543 (1987)). This had been shown by detecting substantial levels of reporter gene proteins or mRNAs in extracts prepared from the leaves, stems, roots and flowers of transgenic plants. As a result, the CaMV35S promoter is widely used in the field of plant genetic engineering. Although the CaMV35S promoter appears to be a strong, constitutive promoter in assays involving cell extracts, detailed histological analysis of reporter gene products detectable at the cell and tissue level shows a rather high degree of variability of expression of the gene products in tissues of plants.
CaMV is a caulimovirus, a subgroup of pararetroviruses that has icosahedral capsids and infects only dicots, although the CaMV35S promoter is a strong promoter in monocots. Sugarcane bacilliform virus (ScBV), Commelina yellow mottle virus (CoYMV) and rice tungro bacilliform virus (RTBV) are badnaviruses, a subgroup of pararetroviruses that have bacilliform capsids and infect mainly monocots. A promoter fragment isolated from CoYMV confers a tissue-specific pattern of expression that is different than the pattern conferred by the CaMV35S promoter. Transformed tobacco plants containing the CoYMV promoter linked to the beta-glucuronidase reporter gene ("GUS"; uidA) showed that while the CoMYV promoter is active in all organs, beta-glucuronidase activity occurs primarily in the phloem, the phloem-associated cells, and the axial parenchyma of roots, stems, leaves, and flowers (Medberry et al., Plant Cell, 4, 185 (1992); Medberry and Olszewski, Plant J., 3, 619 (1993)). In contrast, the CaMV35S promoter is active in most cell types (Medberry et al., Plant Cell, 4, 185 (1992); Medberry and Olszewski, Plant J., 3, 619 (1993)). Moreover, the CoYMV promoter is 30% as active in tobacco suspension cells and up to 25% as active in maize suspension cells compared to a duplicated CaMV35S promoter (Medberry et al., Plant Cell, 4, 185 (1992)).
Transgenic rice containing the RTBV promoter linked to the GUS gene showed strong phloem-specific promoter activity. This was consistent with the expression of this promoter in rice protoplasts. However, the RTBV promoter showed only weak activity in maize protoplasts (Bhattacharyya-Pakrasi et al., Plant J., 4, 71 (1993); Yin et al., Plant J., 7, 969 (1995)). In contrast, the corresponding CaMV promoter shows strong promoter activity in protoplasts and in almost all tissues of transgenic plants (reviewed by Hohn and Futterer, Curr. Opin. Genet. Dev., 2, 90 (1992)).
Thus, what is needed is a highly expressed, constitutive promoter to express transgenes in fertile transgenic monocot and dicot plants.